Hindawi Publishing Corporation

Journal of Immunology Research

Volume 2014, Article ID 938576, 5 pages
http://dx.doi.org/10.1155/2014/938576

Research Article
Unique Cytokine Signature in the Plasma of
Patients with Fibromyalgia

Jamie Sturgill,1,2 Elizabeth McGee,2,3 and Victoria Menzies1,2

1
School of Nursing, Virginia Commonwealth University, Richmond, VA 23298, USA
2
Institute of Women’s Health, Virginia Commonwealth University, Richmond, VA 23298, USA
3
Department of Obstetrics, Gynecology and Reproductive Sciences, University of Vermont, Burlington, VT 05401, USA

Correspondence should be addressed to Jamie Sturgill; sturgilljl@vcu.edu

Received 19 November 2013; Revised 28 January 2014; Accepted 5 February 2014; Published 11 March 2014

Academic Editor: Jong-Young Kwak

Copyright © 2014 Jamie Sturgill et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Fibromyalgia (FMS) is a chronic pain syndrome with a complex but poorly understood pathogenesis affecting approximately 10
million adults in the United States. The lack of a clear etiology of FMS has limited the effective diagnosis and treatment of this
debilitating condition. The objective of this secondary data analysis was to examine plasma cytokine levels in women with FMS
using the Bio-Plex Human Cytokine 17-plex Assay. Post hoc analysis of plasma cytokine levels was performed to evaluate patterns
that were not specified a priori. Upon examination, patients with FMS exhibited a marked reduction in TH 2 cytokines such as IL-4,
IL-5, and IL-13. The finding of this pattern of altered cytokine milieu not only supports the role of inflammation in FMS but also
may lead to more definitive diagnostic tools for clinicians treating FMS. The TH 2 suppression provides strong evidence of immune
dysregulation in patients with FMS.

1. Introduction symptoms of FMS. Thus, it is theoretically plausible that these

Fibromyalgia (FMS) is a chronic pain syndrome in which
pathogenesis is complex and cure is not known. It affects
approximately 10 million adults in the United States with an
estimated 90% of diagnoses being reported in women [1]. The
symptom profile of FMS includes pain, fatigue, and distressed
mood. Sequelae of FMS include physical and psychological
distress, loss of work productivity, reduced quality of life,
and increased use of health resources. Annual expenditures
for the diagnosis and treatment of FMS are estimated at
approximately $20 billion, thus presenting a significant bur-
den to patients, their families, and society [2, 3]. Although the
incidence of FMS is rising, the etiology remains unclear. A
major theory is that inflammatory mediators lead to complex
neuroendocrine aberrations of the hypothalamic-pituitary-
adrenal (HPA) axis [4]. Altered levels of cytokines have been
associated with symptoms of pain, fatigue, and distressed
mood in multiple conditions including painful peripheral
neuropathies, hepatitis C, cardiovascular disease, and cancer
[3, 5, 6]. This symptom profile mimics the representative
nonspecific inflammatory mediators may also contribute
to the symptoms of pain, fatigue, and distressed mood in
FMS. To date, results of studies examining the association
of cytokine alterations with FMS and its symptoms have
been mixed [7–9]. Although researchers have suggested FMS
as being an inflammatory state related to a dysregulated
immune system or altered stress response, the pathophysio-
logical role of cytokines continues to remain unclear [9, 10].
Because there are no diagnostic markers for FMS as well as no
identified etiology for the development of FMS, researchers
are still searching for mechanistic signs to identify those
who already have or those who are at risk for developing
fibromyalgia.
T helper lymphocytes are defined by expressing the cell
surface molecule known as CD4 and are subdivided further
based on the cytokines that they produce. The discovery of
the TH 1 and TH 2 paradigm [11] was a pivotal breakthrough
in the field of immunology. This balance and counterbalance
of inflammatory mediators were delineated and ultimately led
to fundamental additions to the knowledge base of cytokine
2 Journal of Immunology Research
biology we understand today. Although TH subsets have
expanded far beyond the initial discovery to include TH 17,
TH 9, TFH and others probably yet to be discovered, we can
still use the TH 1 and TH 2 paradigm to better understand
inflammation at both the bench and bedside. TH 1 immune
responses are historically associated with antitumor and
antiviral responses, whereas TH 2 are associated with humoral
immune responses. However, today these designations are
being expanded to include other disease states. For example
these helper T cell derived cytokines are being examined in
disease states such as schizophrenia [12], depression [13], and
chronic pain [14].
The purpose of the secondary data analysis in this study
was to examine cytokine profiles in women in diagnosed with
FMS and to determine if relationships existed among the
secreted cytokines detected in the plasma and to determine
if any unique cytokine patterns that emerge correlate with
disease symptoms.
2. Patients and Methods
2.1. Patients. Two separate studies were conducted: one pre-
liminary and one for validation. Both were approved by the
Institutional Review Board of Virginia Commonwealth Uni-
versity. Our preliminary study involved 42 females, whereas
the validation study was comprised of 63 females. Inclusion
criteria included age ≥18, female, diagnosis of FMS as defined
by the 1990 American College of Rheumatology (ACR) cri-
teria, no known major psychiatric or neurological conditions
that would interfere with study participation, and an ability to
understand and sign the consent form. The 1990 ACR criteria
for fibromyalgia require that an individual has both a history
of chronic widespread musculoskeletal pain (more than 3
months) and the finding of 11 of 18 possible tender points
upon physical examination [15]. Both studies were completed
prior to the publication of the 2010 revised FMS diagnostic
criteria [16]. Exclusion criteria included presence of other
systemic rheumatologic conditions, being immunocompro-
mised (e.g., diagnosis of HIV/AIDs), receiving corticosteroid
treatments, being treated for cancer, and/or being pregnant.
Self-reported diagnosis of FMS was confirmed by the partic-
ipant’s primary physician or rheumatologist.
2.2. Questionnaires. In both studies, study participants
completed self-report form to collect data regarding age,
race/ethnicity, marital status, length of time since diagnosis
of FMS, socioeconomic status and psychiatric, medical and
medication history. Stress was measured using the Perceived
Stress Scale (PSS). The 10-item PSS measures the degree to
which the individual perceived events in her life over the
previous month to be stressful. The scale has an internal
reliability of 0.78 and demonstrated construct validity [17].
Pain was measuredusing the Brief Pain Inventory (BPI)
Short Form [18]. The BPI assesses pain severity (BPI-S) and
pain interference (BPI-I) using 0–10 numeric scales for item
rating; higher scores indicate increased pain/interference.
Pain severity indicates the intensity of the pain experienced,
while pain interference measures the degree to which pain
Table 1: Patient demographics.
Number of patients enrolled N = 42
Female N = 42 (100%)
Race
N = 17 African American (41.9%)
N = 25 Caucasian (58.1%)
Age Average = 48.7 (Range 26–75)
interferes with activities of daily living. In widespread testing,
the Cronbach’s alpha reliability ranges from 0.71 to 0.91 [19].
Fatigue was measuredusing the Brief Fatigue Inventory (BFI),
a simple, 9-item scale that taps into a single dimension of
fatigue severity and the interference fatigue creates in daily
life. A score of 7 or higher indicates severe fatigue [20]. The
BFI has demonstrated excellent reliability in clinical trials,
ranging from 0.82 to 0.97 [19]. Depression was measured
using the Center for Epidemiological Studies Depression
Scale (CES-D). The CES-D is a 20-item self-report instru-
ment comprised of four factors assessing cognitive and
affective components of depression. This instrument has very
good construct validity, internal consistency, and test-retest
reliability [21].
2.3. Immunological Assays. Blood samples were collected
into heparinized vacutainer tubes for measuring immune
markers. Blood was centrifuged for separation of plasma, and
all specimens were aliquoted immediately, frozen, and stored
at −80∘ until all samples were collected. All samples were
assayed together to reduce interassay variability.
Plasma levels of cytokines such as interleukin (IL) 1beta
(IL-1𝛽), IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p70, IL-13, IL-
17, G-CSF, GM-CSF, IFN-𝛾, and TNF as well as chemokines
such as CXCL8 (IL-8), CCL2 (MCP1), and CCL4 (MIP1𝛽)
were analyzed using the 17-plex Bio-Rad (Bio-Rad; Hercules,
CA) cytokine, chemokine, and growth factor assay kit per
manufacturer’s protocol.
2.4. Data Analysis. All data are presented as the mean +/−
standard error of the mean (SEM). All secondary analysis was
performed using SigmaPlot software.
3. Results
3.1. Initial Study. The patient demographics can be found
in Table 1. Post hoc analysis of plasma cytokine levels was
performed to determine if patterns appeared that were not
specified a priori. Given the fact that the patients were of
only two races, analysis was first performed to determine
if differences existed in the cytokine levels of Caucasian
women with FMS versus African American women with
FMS. Using Mann-Whitney 𝑈 tests, 16 of the 17 cytokines
assayed displayed no statistical difference among race (data
not shown) and were thus used for further analysis. Power
analysis was performed to ensure a 95% confidence level
with a confidence interval of 15%. Results from this analysis
indicated that an appropriate 𝑁 for cytokines would be
equal to or greater than 21. We then eliminated three more
Journal of Immunology Research 3

Table 2: Cytokine measured. Table 4: Cytokines validated.

Cytokine/chemokine Average ± SEM Normal range Cytokine/chemokine Average ± SEM

CCL2 38.0 ± 3.1 50.0–298.8 IL-4 1.8 ± 1.1
CXCL8 14.5 ± 10.8 6.4–20.4 IL-5 1.0 ± 0.4
IL-l𝛽 4.3 ± 3.4 0.0–1.2 IL-13 4.4 ± 1.4
IL-4 0.6 ± 0.1 5.5–12.5 GCSF 12.4 ± 2.4
IL-5 1.2 ± 0.1 6.0–44.5
IL-6 9.1 ± 2.9 11.0–17.0 20
IL-7 9.5 ± 0.8 0.0–22.0
IL-12p70 5.7 ± 0.6 1.7–2.5
IL-13 2.8 ± 0.3 12.0–47.9 15
GCSF 89.8 ± 6.1 0.0–34.4
GMCSF 26.9 ± 5.9 2.0–48.0

Pain
10
IFN𝛾 32.9 ± 6.5 1.2–25.0
TNF 16.4 ± 2.1 2.1–46.0 P = 0.07
5
Table 3: Patient demographic.

Number of patients enrolled N = 63

Female N = 63 (100%)
N = 18 African American (28.6%)
Race
N = 45 Caucasian (71.4%)
Age Average = 47 (Range 18–71)
cytokines for further analysis given that the 𝑁 of patients with
detectable levels was below 21. These stringency criteria left
13 cytokines and chemokines for further examination. The
values for our FMS patients are presented in Table 2.
Due to the fact that at study initiation normal controls
were not collected, we used plasma values reported in the
literature [22–30]. While we acknowledge this weakness, we
are confident that the analyses are still powerful and will
warrant further examination into cytokine deviations present
in FMS. In order to move forward in the study, we only looked
at cytokines that were at least 2X greater in difference than
2SEMs of the mean. Thus, IL-4, IL-5, IL-13, and GCSF were
further examined. Upon further scrutiny of the remaining
4 cytokines, a stark pattern began to emerge in that these
cytokines are associated with TH 2 immunity.
3.2. Validation Study. To confirm these findings, a secondary
set of FMS were analyzed. These patients were recruited
for a different study and thus were completely independent
of the subjects enrolled in the initial study. The patient
demographic can be found in Table 3. Given the drastic
differences observed in the original data set for IL-4, IL-5, IL-
13, and GCSF, these cytokines were further scrutinized in the
independent validation data set. Using the aforementioned
criteria, we confirmed that IL-4, IL-5, and IL-13 are indeed
suppressed in patients with FMS (Table 4).
3.3. Correlation Analysis. Upon the observation that patients
with FMS had suppression in TH 2 cytokines, we began to
explore the potential relationship between TH 2 immunity
and the psychometrics obtained from the individuals. While
0
0 10 20 30 40 50
TH 2 cytokines
Figure 1: Correlation of pain and TH 2 cytokines. A total TH 2 value
was determined by adding plasma values of IL-4, IL-5, and IL-13. A
total pain value was determined adding BPI-I and BPI-S using the
Brief Pain Inventory scale as described in the material and methods.
A Spearman’s Correlation was performed on the two values using
SigmaPlot and data is shown. Correlation coefficient −0.391, 𝑃 value
0.07.
no correlations proved significant among cytokine levels and
fatigue, depression, or stress (data not shown), there is a trend
towards significance when we compared TH 2 cytokine levels
and pain (𝑃 = 0.07, Spearman’s) as shown in Figure 1.
4. Discussion
Although not classified as an immune disease by nature, our
group as well as others has reported cytokine and immune
alterations in patients with FMS [3, 7, 9, 15, 23]. Given the
fact that disease etiology is still uncertain, further research is
needed in the field to help uncover exact disease pathology
in hopes to provide better therapeutic options the millions
of FMS patients worldwide. The purpose of this analysis
was to examine cytokine alterations in patients with FMS
that were not determined a priori. Comparing the observed
cytokines in the plasma of these patients we noticed a stark
decrease in the amount of TH 2 cytokines produced (IL-4,
IL-5, and IL-13) as those values reported by other groups
in the literature. We extended our analyses to include a
secondary, independent data set and once again this unique
cytokine signature was observed. To examine a potential
underlying cause for the TH 2 suppression, we then correlated
cytokine levels to pain, stress, fatigue, and depression which
are all symptoms shared in the FMS spectrum. When pain
and TH 2 levels were compared we observed a trend that
4 Journal of Immunology Research
approached statistical significance. Interleukin 4, the classic
TH 2 cytokine has been shown to have both anti-inflammatory
and analgesic properties in murine models of mechanical [31]
as well as having lower gene expression and serum levels in
patients with widespread pain syndromes [6]. IL-13 exhibits
analgesic properties in a murine model of L. major infection
[32], whereas little is reported in regard to IL-5’s ability to
combat pain. Thus, our preliminary findings suggest that
further research into the TH 1-TH 2 imbalance in FMS and its
implication in pain are certainly warranted.
Conflict of Interests
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Acknowledgments
Components of the research were supported by the National
Institute of Nursing Research through Grant no. P20
NR008988 (N. McCain, PI) and Grant no. P30 NR011403 (MJ
Grap, PI).
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